The present invention is related to methods of testing samples for environmental contaminants, in particular, for xenobiotic contaminants, using zinc fingers.
The chemical toxic load in the environment has steadily increased over the past 50 years due to increased industrialization, manufacturing, and technology advancement. Most of the chemical agents, including xenobiotics or environmental chemicals of a non-physiological origin, pose major health risks. Therefore, the detection and monitoring of such potentially dangerous chemical toxins is becoming increasingly important and necessary.
It is well known that environmental chemicals of a non-physiological origin (xenobiotics), as well as chemicals which mimic physiological compounds can have deleterious effects on human biology. As used herein, the term "xenobiotic" refers to those elements or compounds which are potentially toxic to biological systems, including elements and compounds having no physiological or biological origin or utility and those elements and compounds which may have biological origin or utility but which at some concentration have biologically inhibitory effects. Because of rapid industrialization and the accompanying waste generation and land reclamation, the detection and monitoring of harmful pollutants is becoming increasingly important and necessary.
The xenobiotic metal cadmium, for example, is believed to be an environmental factor and/or contributor to a variety of human disorders, such as carcinogenesis, teratogenesis, organ failure (kidney), neurological disorders, and immunosuppression. With respect to DNA transcription events, cadmium has been shown to bind chromatin and induce jun and myc oncogene expression. The striking effect of cadmium on TFIIIA-type zinc fingers, as discussed herein below, also points to a role for cadmium in the alteration of gene expression in vertebrates. Also, aluminum is one of the most abundant elements in the earth's crust and is a known neurotoxin. It has also been implicated in the etiology of Alzheimer's disease (AD) and other neuronal disorders.
Based upon these two xenobiotics alone, the sites and resources that require regular biomonitoring are almost limitless.
Various approaches for xenobiotic analyses exist, including classical chemical and living animal studies. Chemical analyses usually involve atomic absorption spectroscopy for metals, and gas chromatography-mass spectroscopy for analyzing organics and inorganics. Rodent systems are the most popular living animals for such analysis. Unfortunately, these chemical and biological studies are time-consuming and quite expensive, thereby negating large scale use which is necessary for extensive detection and biomonitoring. Classical methods of detection may provide information about the concentrations environmental pollutants, but they do not provide direct information on the biological targets or harmful consequences of such agents on vertebrates. Biomonitors and biomarkers can provide such qualitative information, however, most involve fairly complicated, expensive, and time-consuming procedures which typically use living animals or cells.
Because of these unwieldy concerns for pollution analyses with classical chemical and biological systems, other biomonitoring approaches and systems have been developed. One such biomarker approach analyzes DNA adduct formation which results from human exposure to polycyclic aromatic hydrocarbons (Mumford et al., 1993, Environ. Health Pers., 99:83). The methodology for adduct analysis utilizes both fluorescence and immunological assays in a complicated and time consuming process. Two other biomonitor approaches, Microtox.TM. and Artoxkit.TM. pollution analysis systems have been developed. The Microtox.TM. test utilizes the marine bacterium Photobacterium phosphoreum which radiates bioluminescence under appropriate conditions (Microbics Corporation, 1982, Manual 555880-R1, Carlsbad, Calif.). The bacterium is exposed to various concentrations of a suspected pollutant and any reduction in bioluminescence is measured with a luminometer. The Artoxkit.TM. test utilizes larvae of the brine shrimp Artemia salina and assays the effects of pollutants on larvae motion (Persoone and Wells, 1987, Artemia Research and Its Applications, Vol. 1-Morphology, Genetics, Strain Characterization, Toxicology, Universa Press, Wetteren, Belgium).
Recently, a study demonstrated that neither of the Microtox.TM. or Artoxkit.TM. test kits were as sensitive in detecting herbicides as with a traditional algae assay system (Gaggi et al., 1995, Environ. Toxicol. Chem., 14:1065). The algae test system, however, requires sterile conditions and extensive cell growth studies. These cellular pollution analysis systems inherently include the additional variable of the relevancy of the physiological process assayed (e.g. bioluminescence) to human biology. Cellular assays, although obviously easier to work with than rodent systems, are relatively cumbersome compared with an in vittro system which is able to inexpensively assay a large number of samples in a relatively short period of time.
In 1983, it was discovered that cysteine-rich eukaryotic regulatory proteins contain zinc-binding domains and require the zinc ion for function (Hanas et al., J.Biol.Chem., 258:14120). These zinc binding domains were subsequently termed "zinc fingers."A eukaryotic regulatory protein discovered to contain zinc was transcription factor IIIA (TFIIIA), a protein which regulates ribosome synthesis. Each of nine zinc fingers in this protein contains two cysteine (Cys) and two histidine (His) amino acids which bind to a zinc ion.
The Zn.sup.2+ ions hold the structure together, since their removal results in unfolding of TFIIIA and concomitant loss of specific DNA binding ability. Crystallographic analysis of TFIIIA-type Cys.sub.2 His.sub.2 zinc finger domains bound to DNA revealed compact finger domains wrapped around the major groove. The centrally located Zn.sup.2+ ion in each finger was coordinated by the two Cys residues in an antiparallel .beta.-sheet and by the two His residues located on the same face of an .alpha.-helix. Residues in the .alpha.-helix interact specifically via hydrogen bonds with base pairs in the DNA, whereas other amino acids throughout the domain make ionic contacts on DNA phosphates. Mutagenesis of TFIIIA revealed the necessity for all four metal coordinating residues, as well as the integrity of interfinger linker regions, for specific DNA binding. Proteins containing TFIIIA-type zinc finger domains (Cys.sub.2 His.sub.2) are now known to number in the thousands in vertebrates and constitute the largest known superfamily of proteins in all organisms. TFIIIA-type zinc finger proteins regulate a multitude of processes, including embryogenesis and oncogenesis.
The steroid hormone receptor superfamily comprises another large group of cysteine-rich zinc finger transcription factors which translocate into the nucleus upon hormone binding. These proteins activate expression from enhancer regions of a number of hormone responsive genes. Unlike the TFIIIA superfamily, the DNA binding domains of hormone receptors always contain just two Cys.sub.2 Cys.sub.2 zinc fingers. The first finger and linker region comprise an .alpha.-helix and make specific DNA contacts in the DNA major groove, whereas the second finger is involved in protein--protein interactions forming the active receptor dimer. Because of the cysteine-rich nature of the DNA binding domains of steroid hormone receptors, studies have examined the effects of metals other than zinc on the structure and function of hormone receptors. Such studies have added significance since a number of metal ions, including xenobiotic ions, are believed to have etiological roles in carcinogenesis and other disease processes. For example, with respect to the estrogen receptor (ER), one study indicated that 1 mM Cd.sup.2+ could inhibit DNA binding by activated (hormone-bound) receptors and 0.1 mM Cd.sup.2+ could inhibit initial binding of hormone to receptor (S. S Simons, P. K Chakraborti and A. H. Cavanaugh, J. Biol. Chem., 1990, 265, pp. 1938-1945). Another study demonstrated that Cd.sup.2+ could replace zinc in the ER and suffice for DNA binding, although some functional variation was observed (P. F. Predki and B. Sakar, J. Biol. Chem., 1992, 267, pp. 5842-5846).
Amino acids between the second metal-coordinating Cys residue and the second metal-coordinating His residue bind DNA in a sequence-dependent manner. The nine zinc fingers of TFIIIA bind the internal control region (ICR) of the 5S RNA gene (nucleotides +43 to +96). The fingers appear to bind in groups of three with the N-terminal group binding to the 3' C-box of the ICR, the middle group binding in M-box, and the C-terminal three binding the A-box. Binding of the middle and C-terminal finger groups is dependent upon initial binding of the N-terminal group. Mutations in zinc-binding residues in the N-terminal fingers abolish TFIIIA binding whereas similar mutations in the more C-terminal fingers still allow binding by the N-terminal finger group. Zinc ions can be removed from TFIIIA, leading to unfolding of the protein and loss of specific DNA binding ability. Proteins in the TFIIIA superfamily differ in their number of zinc fingers and also in their amino acid sequences, thereby leading to differing DNA binding specificities. Prominent members of the TFIIIA superfamily (e.g. EGR1 family) control cell growth, differentiation, and embryogenesis. Alterations in several of these Cys.sub.2 His.sub.2 zinc finger proteins cause tumorigenesis and teratogenesis. For example, mutations in one such protein, Wilms' tumor suppressor, cause a pediatric kidney tumor. The functions of the majority of the TFIIIA superfamily, however, are unknown.
Many other cysteine-rich factors known in the art contain zinc fingers although of slightly different structure than that found in TFIIIA. All the different kinds of cysteine-rich zinc finger proteins number in the thousands in vertebrates and are known to constitute the majority of the regulators of gene expression and signaling. Biochemically, cysteine-rich zinc finger domains perform specific interactions with DNA, RNA, and proteins. Because zinc ions are not tightly bound to the cysteine residues in such proteins, and because of the high reactivity of cysteine in the reduced form, zinc finger proteins are likely to be susceptible to conditions which interfere with zinc binding leading to inhibition and/or alteration of the zinc finger functional ability, thereby resulting in deleterious effects on cell function and organ physiology. In addition, the agents which harm cysteine-rich zinc finger proteins may also affect other cellular proteins, including those containing critical cysteine residues and other nucleophilic amino acid side chains like imidazole, hydroxyl, amides, carboxyl, and amines. Because these effects pose major health risks, it is important and necessary to develop rapid assay methods to detect and monitor xenobiotic agents in the environment which have the potential to harm zinc finger proteins.